The world demand for paper is expected to increase nearly 50% by the year 2010 (McNutt and Rennel, Pulp Paper Intern 39: 48 (1997)). The United States' forest products industry faces a great challenge in order to keep pace with the growing demand for paper. This challenge is made greater by the decreasing availability of a forest land-base, resulting from environmental restrictions and urban growth, from which to harvest timber resources. Additionally, valuable wood resources are lost to the environmental stresses and biotic diseases. Consequently, the push to secure a renewable and sustainable source of raw material for paper and other wood related products has become an important priority for the forest products industry.
Current forestry related research and development is focused on creating sustainable fiber farms or tree plantations. Farming trees with elite germplasms will increase growth rates and yields of wood per acre. However, creating improved tree stock requires the ability to identify and generate genetically superior trees and a way to propagate such superior trees without diluting their genetic quotient.
A. Breeding and Selection
Addressing the need to propagate genetically superior trees without genetic diminution demands full research attention. Traditional methods of tree propagation relied on selected breeding programs to achieve genetic gain, i.e., the development of a strain, sub-strain, or line having any heritable and economically valuable characteristic or combination of characteristics not found in the parents. Based on the results of progeny tests, superior maternal trees are selected and used in “seed orchards” for mass production of genetically improved seed. The genetic gain in such an open-pollinated sexual propagation strategy is, however, limited by the breeder's inability to control the paternal parent. Additional gains can also be achieved by control-pollination of the maternal tree with pollen from individual trees whose progeny have demonstrated superior growth characteristics. Nevertheless, even under controlled conditions where both parents of each seed are the same, sexual propagation results in a “family” of seeds, i.e., siblings, comprised of many different genetic combinations. As not all genotype combinations are favorable, the genetic gain in any particular progeny is frequently offset and obscured by the genetic variation among sibling seeds and those seedlings retaining undesirable or previously masked pre-cross traits.
In addition to inherent genetic limitations of a traditional breeding programs, large-scale production of control pollinated seeds is also expensive. Consequently, economic and biological limitations of large-scale seed production has lead the industry to turn towards methods of asexual reproduction, such as grafting, vegetative propagation and micropropagation, as more viable alternatives.
B. Asexual (Clonal) Propagation
Asexual propagation permits the application of very high selection intensity, resulting in the propagation of only those progeny showing a high genetic gain potential. These highly desirable progeny can have unique genetic combinations that result in superior growth and performance characteristics. Thus, with asexual propagation it is possible to genetically select individuals while avoiding a concomitant reduction of genetic gain due to intra-familial variation.
Asexual propagation of trees can be accomplished currently by grafting, vegetative propagation, and micropropagation. Grafting, widely used to propagate select individuals in limited quantities for seed orchard establishment, is not applicable to large-scale production for reforestation. Vegetative propagation, achieved by the rooting of cuttings, and micropropagation by somatic embryogenesis, currently hold the most potential for reforestation of conifers. Although vegetative propagation by rooted cuttings can be achieved in many coniferous species, large-scale production via this method is extremely costly due to difficulties in automating and mechanizing the process, not to mention the need for tremendous quantities of stock tissue. This propagation method is still further limited by the fact that the rooting potential of stock plants decrease with time, making it difficult to serially propagate from select genotypes over extended periods of time.
Micropropagation is the most promising method of asexual propagation for mass plantings. This process involves the production of somatic embryos in vitro from minute pieces of plant tissue or individual cells. The embryos are referred to as somatic because they are derived from the somatic (vegetative) tissue, rather than from the sexual process. Both vegetative propagation and micropropagation have the potential to capture all genetic gain of highly desirable genotypes. However, unlike conventional vegetative propagation methods, somatic embryogenesis is amenable to automation and mechanization, making it highly desirable for large-scale production of planting stock for reforestation. Moreover, somatic embryogenesis is particularly amenable to high intensity selection of a large number of clones. These advantages are compounded by the ability to safely preserve somatic embryogenic cultures in liquid nitrogen for long-term storage. Consequently, long-term cryogenic preservation offers immense advantages over other vegetative propagation systems that attempt to maintain the juvenility of stock plants. Techniques for somatic embryogenesis in a wide variety of plant species are well known in the art; exemplary methods for performing somatic embryogenesis in conifers are taught in U.S. Pat. Nos. 5,036,007; 5,236,841; 5,294,549; 5,413,930; 5,491,090; 5,506,136; 5,563,061; 5,677,185; 5,731,203; 5,731,204; and 5,856,191, herein incorporated by reference in their entirety.
Thus, somatic embryogenesis has great potential for clonal production of conifer embryos to meet the increased demands of the pulp and paper industry. Assessment of embryo quality, however, needs improvement. The process of creating better tree stock begins with understanding the process of tree development from embryogenesis through full maturation.
In general, plant tissue culture is the broad science of growing plant tissues on or in a nutrient medium containing minerals, sugars, vitamins and plant hormones. By adjusting the composition of the media, cultured tissues can be induced to grow or differentiate into specific cell types or organs. “Somatic embryogenesis” is a type of plant tissue culture where a piece of a donor plant is excised, cultured and induced to form multiple embryos. An embryo is a discrete mass of cells with a well-defined structure that is capable of growing into a whole plant.
The methods generally in use for somatic embryogenesis today involve several steps. Prior to the tissue culture process, a suitable “explant” is harvested. A typical explant in conifer somatic embryogenesis is the “megagametophyte”, a haploid nutritive tissue of the conifer seed, which is extracted from the ovule of a pollinated female cone. This ovule contains single or multiple zygotic seed embryos. In the seeds of many coniferous species, one or more genetically unique embryos naturally undergo a process called cleavage polyembryony, where a zygotic embryo grows and divides to form a small clones of embryos.
The first step in somatic embryogenesis is the initiation step. The explant is placed on a suitable media. When the explant is an ovule, a process called extrusion occurs. Extrusion involves the emergence or expulsion of a zygotic embryo or multiple embryos and embryogenic tissue out of the megagametophyte. If culture conditions are suitable, initiation proceeds and the extruded embryo or embryos undergo the process of cleavage polyembryony. This results in the formation of early stage somatic embryos in a glossy, mucilaginous mass.
After embryogenic cultures are initiated, the somatic embryos are transferred to a second medium with an appropriate composition of plant hormones and other factors to induce the somatic embryos to multiply. In the multiplication stage, cultures can double up to 2–6 times in one week. Once large numbers of embryos are obtained in the multiplication stage, the embryos are moved to a development and maturation medium. Here, the correct balance of plant hormones and other factors will induce the early-stage embryos to mature into late stage embryos. Following the maturation and development stage, embryos are germinated to form small seedlings. These seedlings are then acclimated for survival outside of the culture vessel. After acclimation, the seedlings are ready for planting.
The relative ability to propagate plants by somatic embryogenesis can vary greatly between species. Among conifers, for example, spruce (Picea) species and Douglas fir are easily propagated, while Pinus species are much more difficult. Many Pinus species, including Loblolly pine (Pinus taeda), do not readily initiate embryonic cultures. Typical initiation frequencies between 1% and 12% are reported for various Pinus species (Becwar et al., For. Sci. p1–18 (1988), Jain et al., Plant Sci. 65:233–241 (1989), Becwar et al., Can. J. For. Res. 20:810 (1990), Li and Huang, J. Tissue Cult. Assoc. 32:129 (1996)). Laine and David, (Plant Sci. 69:215 (1990)), however, were able to obtain high frequencies of initiation (up to 59%) in Pinus caribaea, suggesting that not all Pinus species are recalcitrant. Also, one earlier report described initiation frequencies of 54% in White pine (Pinus strobus). Finer et al., Plant Cell Rep. 8:203 (1989). However, other workers were not able to duplicate this success. Michler et al., Plant Sci. 77:111 (1991). The results in the literature demonstrate the recalcitrance of Pinus species, especially Loblolly pine, in regeneration by somatic embryogenesis.
Nevertheless, once this process is understood from the standpoint of developmental genetics, breeders will then have the appropriate tools to monitor, intervene, and improve both the regeneration frequency and the overall quality of tree stock through genetic engineering. For example, both environmental requirements and responsiveness of a developing embryo change as the embryo passes various developmental milestones. Consequently, accurate and timely knowledge of the developmental stage of an embryonic culture would allow the skilled practitioner to beneficially adjust the growth media components and other environmental factors to achieve optimal embryo survival, growth, and maturation. In addition, an understanding of developmentally regulated genes would allow for early selection of advantageous clones and provide tools for developmentally regulated transgenic expression systems.
Currently, a reasonable determination of the precise developmental stage of an embryo requires a practiced, physical familiarity with the morphological appearance of embryos at different stages, which is further complicated by the presence of morphological variations between species. Consequently, visual determination is performed best by experts in the field. Thus, there is a need in the art for a staging method which can be reliably practiced by the ordinary practitioner. The current invention will allow one to stage embryos based on a relational database system profiling gene expression patterns instead of physical morphological differences, thereby permitting one less skilled in the art of visual staging to biologically determine the stages of embryogenesis.
The traditional morphological staging method provides only a crude indication of the underlying biochemical condition or state of an embryo. This level of information is insufficient for refining culture conditions, including media formulations, or for selecting potentially advantageous embryo clones for further development. Thus, there is a need in the art for a more sensitive staging method that precisely defines the physiological age, health, growth requirements, and potential fitness of a particular embryo. The current invention will allow definitive staging significantly beyond that currently practiced in the art, and provides a detailed analysis of the biochemical state and potential fitness of an embryo by comparison to developed relational database profiles.
Visual staging methods depend on morphological markers to assign a numerical stage of 1–9 to an embryo. Nevertheless, it is well accepted that visually undetectable developmental changes occur in an embryo after it reaches stage 9. The current invention is particularly useful in providing means for monitoring and evaluating the developmental state of these older embryos, as genetic responses occur and are detectable up to and through an adult tree's life.
There further exists in the art a need for information regarding the proteins, genes, and gene expression patterns in plant embryo development, as well as a more thorough understanding of how this information relates to the physiology, developmental potential, and genetic quotient of a plant embryo. The relational database system provides a platform for which to monitor individual gene expression levels during embryo development while directly correlating expression with, for example, environmental conditions, age, and embryo fitness, as well as the protein identification achieved by BLAST searches of publicly available databases (i.e., GenBank) for desirable genes. Accordingly, the present invention therefore provides the additional ability to correlate the direct, global gene expression response within the embryo system to a typically non-expressing gene driven by a stage-specific promoter.